Listeria monocytogenes (Lm) is an attractive bacterial vector to elicit T cell immunity to HIV because it specifically infects key antigen presenting cells (APCs) and because natural infection originates at the mucosa. Immunization with recombinant Lm protects mice from LCMV, influenza and tumor inoculation. Lm expressing HIV gag elicits sustained high levels of gag-specific CTL in mice. Since Lm can cause serious disease in immuno-compromised hosts, a more highly attenuated strain that requires D-Ala for viability and is attenuated at least 105 fold in mice was produced. The hyper-attenuated bacteria expressing HIV-1 gag (Lmdd-gag) are efficient as wild-type recombinants at stimulating gag-specific murine CTL in vivo when administered with D-Ala. Oral or systemic immunization with Lmdd-gag protects mice in a vaccinia challenge model and induces mucosal CTL in mouse gut-associated lymphoid tissues, even after systemic immunization. Immunization of newborn mice protects against heterologous mucosal or systemic challenge as adults. Preliminary studies in 2 macaques suggest that Lmdd-gag is safe and stimulates HIV-specific immune responses, even when administered orally. Attenuated nef and gag constructs boost human CTL responses in vitro. Because Lmdd is an attractive candidate vaccine vector to induce T cell immunity to HIV in humans, this program will perform pre-clinical studies in rhesus macaques to establish the proof of principle that Lmdd-vectored vaccines encoding SHIV antigens are immunogenic and provide protection from SHIV challenge in rhesus macaques. The focus of the program will be on developing Lmdd vectors as oral vaccines to optimize mucosal immunity at the predominant sites of HIV transmission. Lmdd vectors will be constructed expressing SHIV genes for macaque challenge experiments and expressing consensus HIV clad B, B' and C genes suitable for later-human studies. Mouse experiments will lay the groundwork for optimizing the immunization route, dose and boosting schedule. Recombinant Lmdd stably expressing SHIV89.6P and clade C R5-using SHIV11578ip gag, nef, env and tat genes will be tested for safety, immunogenicity and protection from challenge in adult rhesus macaques. A pregnant mother/neonatal transfer model in macaques will also be used to test for safety and immunogenicity in an animal model that utilizes the animals most vulnerable to Listeria toxicity.